Influenza virus is the causative agent of a highly contagious respiratory illness, commonly named “flu”, which affects animals and humans causing public health and economic problems. The influenza virus is an enveloped RNA virus with a segmented genome consisting of single-stranded negative RNA segments. Influenza viruses encompass the three types: influenza A, influenza B and influenza C viruses. Influenza A and B viruses are responsible for human influenza epidemics resulting in the death of over 50 000 people per year (Rossman et al, 2011, Virology, 411(2):229-236). While influenza A viruses infect both humans and a broad variety of animals (birds, pigs, horses, dogs, cats, etc.), the largest natural reservoir being wild aquatic birds, influenza B viruses are predominantly restricted to humans which is partially caused by the inability of B/NS1 protein to counteract the innate immune response of others species (Sridharan et al, 2010, J Biol Chem, 285(11):7852-7856) and influenza C viruses are isolated from humans and pigs.
The type A viruses have a spherical or filamentous shape and have a size of about 80 to 150 nm. The viral envelope, consisting of a lipid bilayer, is derived from the plasma membrane of the host cell. Spicules formed of surface glycoproteins, HA (hemagglutinin) and NA (neuraminidase), the main targets for the host antibodies, are inserted into this envelope. The M2 protein, which is also embedded in the membrane, is an ion channel that functions mainly during decapsidation of the virus. The matrix protein M1 is located on the inner periphery of the virus associated with the lipid bilayer and with the ribonucleoprotein (RNP). It has a fundamental role in the nucleo-cytoplasmic export of RNPs. In the capsid, the vRNA segments possess noncoding 5′ and 3′ ends containing the signals necessary for the transcription, the replication and the encapsidation of the viral genome. The eight vRNA of influenza A viruses called PA (Polymerase Acidic), PB1 (Polymerase Basic protein 1), PB2 (Polymerase Basic protein 2), NP (Nucleoprotein), HA, NA, M and NS (Non-Structural protein) encode one or more proteins by alternative splicing. The PA segment encodes the PA protein; the PB1 segment encodes the PB1, PB1-F2 and PB1-N40 proteins; the NP segment encodes the NP protein; the HA segment encodes the HA protein; the NA segment encodes the NA protein; the M segment encodes the M1 and M2 proteins; the NS segment encodes the nonstructural proteins NS1 and NS2 or NEP (Nuclear Export of vRNPs). The vRNAs are coiled over NP which binds 24 nucleotides per monomer, the polymerase complex binds to the two ends of the RNA molecule, forming an hairpin structure. This complex consists of PB1, PB2 and PA. The RNA, NP and polymerase combination forms the ribonucleoprotein (RNP) complex.
Type B viruses have a glycoprotein in addition to NA called NB which has a type III structure like the protein M2.
Type C viruses have only one multifunctional surface glycoprotein, “hemagglutinin-esterase-fusion protein” (HEF).
Thus, the genome of types A and B viruses contains 8 viral RNA (vRNA) while the genome of the influenza virus type C contains only 7.
Influenza A viruses are also divided into distinct subtypes according to the nature of the surface viral glycoproteins, i.e. currently hemagglutinin (HA) (H1 to H17) and neuraminidase (NA) (N1 to N9).
The discovery by Burnet, in 1936, that influenza virus could grow in embryonated hen's eggs has enabled the study of their properties and has permitted the development of inactivated vaccines (De Ona et al, 1995, J Clin Microbiol, 33(7):1948-1949). As described by the World Heath Organization (WHO), vaccination is the most effective way to prevent infection. Fortunately, safe and effective vaccines have been available for more than 70 years. The seasonal flu vaccine contains different influenza types and subtypes (A/H1N1, A/H3N2 and B) that are updated twice a year (once for the northern hemisphere and once for the southern) because of antigenic modifications. For this reason, the WHO coordinates a Global Influenza Surveillance Network (GISN) to monitor the epidemiology of influenza viruses. Once the viruses to be included in next seasonal vaccine have been determined, candidate high-growth seed virus strains must be prepared by WHO Collaborating Centers like the New York Medical College (NYMC, US), the National Institute for Biological Standards and Control (NIBSC, UK), the CSL group (Australia) and the National Institute for Infectious Diseases (NIID, Japon) (Gerdil et al, 2003, Vaccine, 21(16):1776-1779). Vaccine strains are then amplified on eggs, MDCK or Vero cell lines by manufacturers (Koudstaal et al, 2009, Vaccine, 27(19):2588-2593). Currently, MDCK (Tree et al, 2001, Vaccine, 19(25-26):3444-3450), Vero (Kistner et al, 1998, Vaccine, 16(9-10):960-968) and PER.C6® (Pau et al, 2001, Vaccine, 19(17-19):2716-2721) are the three cell lines which may meet the regulatory requirements and have been shown to successfully ensure the replication of influenza A and B viruses. All three cell lines have been adapted to grow in serum free media (Coussens et al, 2011, Vaccine, 29(47):8661-8668).
The introduction of the influenza viruses into the cells (first step of infection) occurs through specific interaction between Influenza hemagglutinin (HA) surface protein and specific cell surface receptors. The host cell membrane receptors specific for influenza viruses are made of carbohydrate structures of sialyl lactosamine chains (sialic acid [Sia] alpha2-3/6 galactose [Gal] beta1-4/3 N-acetyglucosamine) (Suzuki et al, 2011, Adv Exp Med Biol, 705:443-452). Human influenza viruses preferentially bind to cellular receptors containing a Sia2-6Gal linkage, whereas avian viruses preferentially bind to Sia2-3Gal receptors (Coussens et al, 2011, Vaccine, 29(47):8661-8668). When two viruses infect the same cell, different combinations of genomic vRNAs, called reassortants, may arise. This property has been used for the production of influenza A vaccines to combine the antigenic properties of HA and neuraminidase (NA) proteins of target circulating viruses with the favourable growth characteristics (internal genes) of an egg-adapted virus, called A/Puerto Rico/8/34 (PR8) (H1N1). Unfortunately, success in deriving the desired high yielding virus is unpredictable. In addition, some strains cannot be used if they have been isolated on non-validated cell lines as they are not acceptable by the regulatory authorities as a progenitor vaccine strain (Nicolson et al, 2005, Vaccine, 23(22):2943-2952). With respect to influenza type B viruses until very recently no B virus having the growth characteristics of A/PR/8/34 (H1N1) virus has been identified. Therefore, the epidemic circulating (or seasonal) B virus was used directly to infect embryonated hen's eggs and several passages were needed to improve the yield of B vaccine strains (Iwatsuki-Horimoto et al, 2008, Virus Res, 135(1):161-165).
Since 1999, significant improvements in terms of speed and safety were achieved thanks to plasmid-based reverse genetics technology which allows the generation of infectious influenza viruses entirely from cloned viral cDNA (Fodor et al, 1999, J Virol, 73(11):9679-9682). Different systems were developed based on a set of plasmids capable of inducing the expression of the eight vRNAs and at least the polymerase protein complex and the nucleoprotein (NP) required for the transcription. The polymerase protein complex and NP can also be expressed either by transfection of four additional plasmids or by the use of plasmids with bidirectional promoters that allow both vRNA and mRNA synthesis through RNA polymerase I (POL 1) and II (POL 2) (Jackson et al, 2011, J Gen Virol, 92(Pt1):1-17) respectively. The total number of plasmids transfected can vary from 16 (Neuman et al, 1999, Proc Natl Acad Sci USA, 96(16):9345-9350), or 12 (Fodor et al, 1999, J Virol, 73(11):9679-9682) to 8 (Hoffmann et al, 2002, Vaccine, 20(25-26):3165-3170), depending if the strategy is unidirectional or bidirectional, and from 3 (Neumann et al, 2005, Proc Natl Acad Sci USA, 102(46):16825-16829) to 1 (Zhang et al, 2009, J Virol, 83(18):9296-9303) if plasmid(s) encode(s) several vRNA.
Current reverse genetics systems are based on the use of PER.C6® (Koudstaal et al, 2009, Vaccine, 27(19):2588-2593), CEP (Chicken Embryo Primary) cells or Chicken Embryonic Fibroblasts (CEF) (Zhang et al, 2009, J Virol, 83(18):9296-9303), 293T cells alone (Neuman et al, 1999, Proc Natl Acad Sci USA, 96(16):9345-9350) or with further amplification on MDCK (Hoffmann et al, 2002, Vaccine, 20(25-26):3165-3170; Schickli et al, 2001, Philos Trans R Soc Lond Biol Sci, 356(1416):1965-1973), Vero cells alone (Nicolson et al, 2005, Vaccine, 23(22):2943-2952; Neumann et al, 2005, Proc Natl Acad Sci USA, 102(46):16825-16829) or with further amplification on Madin-Darby Bovine Kidney (MDBK) (Fodor et al, 1999, J Virol, 73(11):9679-9682), CEP cells or CEF (Legastelois et al, 2007, Influenza Other Respi Viruses, 1(3):95-104; Whiteley et al, 2007, Influenza Other Respi Viruses, 1(4):157-166).
When a mixture of cell lines is used to produce virus by reverse genetics method, the cell line which can be transfected the most efficiently is considered as the one which is responsible for the generation of infectious influenza viruses, while the other cell lines contribute to the multiplication of the infectious viruses. Since human RNA POL I promoter is generally used in the plasmids that allow the production of influenza vRNAs, human and simian cells are the most appropriate cell lines to be used as transfected cell line in the reverse genetics system. However POL I promoter from canine or chicken origin can also be used in canine or avian cells respectively (Massin et al, 2005, J Virol, 79(21):13811-13816; Murakami et al, 2008, 82(3):1605-1609). On the other hand, the plasmids that allow the production of mRNA encoding viral proteins usually contain a Cytomegalovirus (CMV) or beta actin POLII promoter that can work in any eukaryotic cell (Neuman et al, 1999, Proc Natl Acad Sci USA, 96(16):9345-9350; Schickli et al, 2001, Philos Trans R Soc Lond Biol Sci, 356(1416):1965-1973).
Most of the time, in the above described reverse genetics systems, the cells are usually cultivated in a serum-containing medium to ensure vigorous growth of the different cell types just before transfection. Furthermore, trypsin from porcine origin is also used in the infection medium to sustain viral proliferation after infection. To obtain enough viruses, several amplifications on eggs or cells may also be needed after the first transfection step.
The pandemic A/H1N1 (2009) virus demonstrated the speed with which an influenza A virus can disseminate among the population and illustrated the need for accelerating reassortant production via reverse genetics. Thus, the main challenge is to ensure that high amounts of doses of vaccine are produced in a minimum of time to be distributed all over the world, ideally faster than virus spread.
Conventional approaches used for cloning require restriction enzymes. However restriction sites are often present in different influenza cDNA complementary to vRNA, requiring either the implementation of vector modifications or viral genome mutagenesis. Simplified recombinational approach was developed previously for cloning influenza cDNA complementary to vRNA for reverse genetics purpose (Stech et al, 2008, Nucleic Acid Res, 36(21):e139; Wang et al, 2008, J Virol Methods, 151(1):74-78). Homologous recombination involves a process of breakage and reunion in regions of identical DNA sequences between two DNA molecules to result in new combinations of genetic materiel (Watt et al, 1985, Proc Natl Acad Sci, 82:4768-4772). These previously described recombinational cloning systems are based on a 25 nucleotides recombination cassette comprising the consensus 5′ (Uni13) and 3′ (Uni12) conserved non-coding ends of influenza A segments between human POL I promoter and terminator. They allow the rapid and direct cloning of any influenza A genome. However, since the nucleotide sequences of vRNA 5′ and 3′ non-coding ends of influenza B genomes are different from influenza A virus, influenza B genomes cannot be cloned based on this recombination cassette.
Thus there is also a need to develop a universal approach for cloning RNA virus genomes, and in particular the influenza A, B and C genomes, as quickly and as efficiently as possible.
It is an objective of the present invention to provide useful tools and methods that facilitate and/or accelerate the production of an influenza vaccine in optimized safe conditions, especially when a new circulating influenza virus has been identified and could be responsible for an epidemic or a pandemic flu.
To this effect the subject matter of the invention is relating to new methods for producing a large panel of infectious type A and type B viruses, including reassortant or chimeric viruses, in particular viruses that have been generated by reverse genetics. These methods make easier the manufacturing of influenza virus in more secure conditions. In another aspect, the invention provides a universal recombinant vector that allows the cloning of any type of influenza RNA fragment from type A or B viruses, which proved to be a useful tool to carry out reverse genetic methods.